The long-range goal of this laboratory is to understand the intracellular signaling pathways in hematopoietic cells and how they regulate outcomes such as cell growth/differentiation. This proposal focuses on the adapter protein Shc and the inositol phosphatase SHIP, and how they regulate phosphotyrosine and phospholipid-media ted signaling events. Of the two different phosphotyrosine-interaction domains of Shc (a PTB domain and an SH2 domain), the PTB domain appears to play an important in signaling via several cytokine receptors. The first aim of this proposal specifically addresses the functional significance of the PTB domain in vivo. Our preliminary NMR data and in vitro binding studies indicate that Shc-PTB domain can interact with two ligands, i.e. tyrosine-phosphorylated proteins and phospholipids. Through structure-based site-directed mutagenesis, the role of this domain in membrane localization and the biological functions of Shc are investigated. Since the Shc-PTB domain may represent a prototype "hybrid" domain, and may participate in both phosphotyrosine and phospholipid signaling, these studies could have significant implications in intracellular signaling. Activation of cytokine receptors and antigen receptors in hematopoietic cells lead to association of Shc with a 145kDa phosphoprotein. Recently, this 145kDa protein has been identified as SHIP (SH2 domain- containing inositol- phosphatase). In vitro, SHIP dephosphorylates the 5'-position of phosphoinositide PIP3 (implicated in a variety of signaling pathways) and IP4 (implicated in calcium signaling). The precise function of SHIP and the molecular mechanisms of SHIP function remain largely unknown, although preliminary evidence suggests that SHIP may play a negative regulatory role in signaling. The second aim of this proposal addresses the role of SHIP in cytokine-induced proliferation, and signaling downstream of the T cell receptor. The potential downstream signaling pathways that may be regulated through SHIP, through its action on PIP3 and IP4, are addressed in detail. The third aim addresses the in vivo functional regulation of Shc and SHIP. Preliminary results indicate that Shc can regulate the enzymatic activity of SHIP and that SHIP may also be regulated through its oligomeric state. Based on these, the biological significance of the Shc:SHIP interaction is addressed using several read-outs. The studies proposed on the molecular understanding of Shc and SHIP function could provide important clues to positive and negative signaling events following receptor activation, integration of phosphotyro-sine and phospholipid signaling, and how these ultimately regulate cell growth and differentiation.